The present invention relates to the production of lipid vesicles (liposomes) and, more particularly, to the production of liposomes from long chain alkylammonium fatty acid salts.
Liposome formation is a natural result of the amphipathic nature of the molecules of which they are comprised. Amphipathic molecules are those molecules with distinct regions of the molecule having hydrophilic character and distinct regions of the same molecule having hydrophobic character. When dispersed in water, amphipathic molecules form three types of macro-molecular structure: micelles, hexagonal phase and lipid bilayers. The exact macro-molecular structure which is formed depends on the relative sizes of the hydrophilic and hydrophobic regions of the molecule.
Micelle formation is favored when the cross sectional area of the hydrophilic region of the molecule is greater than that of the hydrophobic part of the molecule. Detergents are examples of such molecules, e.g., sodium palmitate. Detergents contain a hydrocarbon chain (the hydrophobic portion of the molecule) and an ionic base (the hydrophilic portion of the molecule), and act as emulsifying agents to bind water and oil phases. That is, detergents allow oil and water to be broken into tiny droplets suspended or dispersed in water. Particular detergents 1 may be classified as anions (negatively charged at the hydrophilic portion) and may be represented, as shown in FIG. 1, as having a hydrophilic head 2 with a hydrocarbon (hydrophobic) tail 4. FIG. 2 is a representation of a micelle structure 5 formed of a number of detergent molecules due to their hydrophilic/hydrophobic character.
In the opposite conformation, i.e., when the cross sectional area of the hydrophobic region of the molecule is greater than that of the hydrophilic part of the molecule, the formation of hexagonal phase structures is favored, e.g., dimyristoylphosphatidylethanolamine (DMPE). FIG. 4A is a representation of a hexagonal phase structure, sometimes referred to as an inverse micelle.
For molecules in which the cross sectional area of the hydrophilic region of the molecule is slightly less than, or equal to, that of the hydrophobic part of the molecule, such as many phospholipids, the formation of bilayers is favored, e.g., dipalmitoylphosphatidylcholine (DPPC). Phospholipids are an amphipathic type of lipid which contain phosphate, that is, molecules containing one phosphate, a glycerol and one or more fatty acids. FIG. 3 is a simplified representation of a phospholipid molecule 6, including a hydrophilic head 8 (i.e., the phosphate and glycerol) and a hydrophobic tail 10 (i.e., the one or more fatty acids). FIG. 4 is a representation of a phospholipid bilayer 12, where the hydrophobic regions 14 of the phospholipid molecules are caused to turn inward due to the aqueous environment, and the hydrophilic portions 16 face outward. These bilayers are two dimensional sheets in which all of the hydrophobic portions, e.g., acyl side chains, are shielded from interaction with water except those at the ends of the sheet. An energetically unfavorable interaction of the acyl chains with water results in the folding of the bilayers to form three-dimensional, vesicles. These vesicles are referred to as "liposomes".
Liposomes may be formed as a single bilayer enclosing a single aqueous space (small unilamellar vesicles; SUVs) or may be composed of concentric bilayers with many aqueous spaces alternating with the bilayers (multilamellar vesicles; MLVs). Liposomes can be used to encapsulate both hydrophobic and hydrophilic materials. Hydrophobic payloads are typically partitioned within the bilayers whereas hydrophilic payloads are typically trapped within the aqueous compartments. The advantages of using liposomes as a carrier/encapsulation system is that they are stable and can protect their payload from degradation, e.g., by oxygen, digestive enzymes, etc.
For example, U.S. Pat. No. 3,957,971, issued May 15, 1976, discloses liposome-formed moisturizing units which are capable of moisturizing and improving flexibility, plasticity, and softness of keratinous matter, particularly mammalian skin. The liposomes within which the moisturizer is stored include a matrix of a ternary lipid mixture of lecithin, dicetyl phosphate, and a sterol, and include cavities disposed within the liposome. The cavities (lamellar space) contain an humectant, such as sodium pyroglutamate, in an aqueous medium. Moisturizing liposomes are also disclosed therein which function osmotically, serving as traps for water, which may be shared with the keratin constituents as required.
Liposomes also may be used for the timed delivery of a wide variety of materials including pharmaceuticals, cosmetics and nutrients. For example, U.S. Pat. No. 4,016,100, issued Apr. 5, 1977, discloses a method of producing a pharmaceutical composition comprised of an aqueous suspension of an active agent entrapped in a spherule of a phospholipid (liposome) The composition, or drug delivery vehicle, is prepared by dispersing a phospholipid uniformly in water to give an aqueous phospholipid dispersion, adding a medicament to the aqueous dispersion and freezing the thus-obtained aqueous dispersion to entrap the medicament in lipid spherules formed. The frozen dispersion is then thawed to realize an aqueous suspension of spherules having diameters of less than 5.0 microns. The timed release of an active agent is directly related to the amount of active agent trapped in the liposomes. The greater the amount of active agent, the longer the release process lasts.
A goal of the liposome research has been the development of a liposomal delivery system that would deliver its payload not over time as in the '100 patent mentioned above, but on cue, i.e., a controlled release, for example, in a mammalian body. For example, a delivery system that delivers its payload when applied to the skin or when arriving at a tumor. A bulk of the research has been based on admixtures of liposomes and other biological macromolecules such as antibodies and lecithins. Various degrees of success have been achieved with these systems but none have produced a liposome that will release its payload, or not, depending on the prevailing conditions. The invention described herein is just such a liposome: the degree of payload encapsulation may be altered by changes in pH and/or ionic strength of the surrounding medium thereby realizing a triggered delivery system in a form of a liposome.